This proposal outlines experiments to determine both the primary nucleotide sequence and higher levels of organization of the repeated DNA in the centromeric heterochromatin of Drosophila melanogaster. The bulk of the highly-repeated DNA of this organism consists of four homogeneous satellites, each of which separates into complementary strands in alkaline CsCl gradients, a property which makes them ideal for sequence analysis. The frequency and distribution of "mutations" or alterations of the basic repeat sequence of these DNA species will be examined to determine if they are dispersed in a regular or a random fashion. The arrangement of the four classes of highly-repeated DNA relative to one another within the chromosomal DNA will be analyzed using a sequential hybridization technique. DNA molecules containing more than one class of repeated DNA in covalent linkage will be isolated and their frequency in the genome determined. These hybrid DNA molecules containing any two highly-repeated DNA classes will allow an ordering of these sequences within the centromeric heterochromatin. The same procedure will be used to analyze the arrangement of DNA sequences in the more localized region of the nucleolus organizers and region 21 C-D on the polytene map of chromosome 2. A similar technique will also be used to isolate heterochromatic DNA other than the known repeated sequences.